Live Cell Imaging of Bioorthogonally Labelled Proteins Generated With a Single Pyrrolysine tRNA Gene

Abstract

Genetic code expansion technology enables the incorporation of non-canonical amino acids (ncAAs) into proteins expressed in live cells. The ncAA is usually encoded by an in-frame stop codon (e.g., TAG) and the methodology relies on the use of an orthogonal aminoacyl tRNA synthetase and its cognate amber suppressor tRNA; for example, the pyrrolysine synthetase/tRNA Pyl CUA (PylT) pair. In such systems, suppression of the in-frame stop codon by the suppressor tRNA is highly dependent on the intracellular concentration of the tRNA. Therefore, multiple copies of pylT genes are usually encoded in order to improve ncAA incorporation and protein expression level. However, certain applications of genetic code expansion technology in mammalian cells can benefit from the use of minimal, less invasive, expression systems. For example, live-cell imaging applications, where aminoacylated and labeled suppressor tRNA contributes to high background fluorescence. Therefore, we studied the effect of PylT on livecell fluorescence imaging of bioorthogonally-labeled intracellular proteins. We found that in COS7 cells, a decrease in pylT copy number has no measurable effect on protein expression level and cellular concentration of available PylT. Importantly, we found that reducing pylT copy number improves livecell imaging by enhancing signal-to-noise ratio and reducing immobile PylT population. This enabled us to significantly improve live cell imaging of bioorthogonally labeled intracellular proteins, as well as to co-label two proteins in a cell. Our results indicate that the number of encoded pylT genes should be minimized according to the transfected cell line, incorporated ncAA, and the application it is used for.

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

“…GCEtagged GFP-CAAX was expressed in cells at higher levels than GFP-CAAX that carries a TAG codon at the optimized position in GFP (GFP 150TAG -CAAX, Fig. 3a) [19]. Consistently, PM decoration was observed in cells expressing either GCE-tagged GFP-CAAX or GFP 150TAG -CAAX and labeled with SiR-Tet in both the GFP and SiR channels with relatively similar SNR values (Fig.…”

“…cloned into a single expression vector, designed to encode the incorporation of the ncAA bicyclo-nonyne Lysine (BCN-Lys) that bioorthogonally reacts with tetrazineconjugated Fl-dyes ( Fig. 1b and Additional file 1: Figure S1a) [6,18,19]. Labeling potency was initially assessed using α-tubulin as a benchmark and evaluating microtubule (MT) labeling in live mammalian cells in the presence of Silicon-Rhodamine-Tetrazine (SiR-Tet) [6,20].…”

“…nucleolus. To minimize these noise factors, we previously performed careful optimization of all assay components [6,19]. Using our optimized labeling assay conditions, GCE-tag-based labeling was comparable to protein tags in terms of labeling efficiencies and SNR values.…”

Phosphine‐Activated Lysine Analogues for Fast Chemical Control of Protein Subcellular Localization and Protein SUMOylation

“…This approach allowed us to control protein subcellular localization via multiple mechanisms (controlling NLS function and controlling SUMOylation) and to target proteins to different subcellular locations. The methodology demonstrated here is generally applicable to other proteins, cell types, and model organisms . In contrast to light as another fast trigger of protein function, our small‐molecule‐triggered system does not require specialized equipment, is not affected by tissue opaqueness, and does not interfere with fluorescent reporters .…”

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

“…However, despite a consensus among researchers that flagella are not present during biofilm maturation but only in the dispersion stage, reports regarding this are still somewhat constradicting [10][11][12][13][14][15][16] . Therefore, it is necessary to To date, live-cell imaging can be obtained through different approaches, however genetic code expansion, the reassignment of codons and incorporation of an unnatural amino acid (Uaa) into proteins 17 , displays advantages over other methodologies, and is gaining increasing exposure and momentum [18][19][20][21] . Genetic code expansion systems are being constantly improved, expanded and adapted to a growing number of organisms [22][23][24][25] .…”